Nuclear egress of TDP-43 and FUS occurs independently of Exportin-1/CRM1

Helena Ederle,Susanne M Bailer,Christina Funk,Dorothee Dormann,Eva B E Funk,Ralph H Kehlenbach,Saskia Hutten,Claudia Abou-Ajram

doi:10.1038/s41598-018-25007-5

Abstract

TDP-43 and FUS are nuclear proteins with multiple functions in mRNA processing. They play key roles in ALS (amyotrophic lateral sclerosis) and FTD (frontotemporal dementia), where they are partially lost from the nucleus and aggregate in the cytoplasm of neurons and glial cells. Defects in nucleocytoplasmic transport contribute to this pathology, hence nuclear import of both proteins has been studied in detail. However, their nuclear export routes remain poorly characterized and it is unclear whether aberrant nuclear export contributes to TDP-43 or FUS pathology. Here we show that predicted nuclear export signals in TDP-43 and FUS are non-functional and that both proteins are exported independently of the export receptor CRM1/Exportin-1. Silencing of Exportin-5 or the mRNA export factor Aly/REF, as well as mutations that abrogate RNA-binding do not impair export of TDP-43 and FUS. However, artificially enlarging TDP-43 or FUS impairs their nuclear egress, suggesting that they could leave the nucleus by passive diffusion. Finally, we found that inhibition of transcription causes accelerated nuclear egress of TDP-43, suggesting that newly synthesized RNA retains TDP-43 in the nucleus, limiting its egress into the cytoplasm. Our findings implicate reduced nuclear retention as a possible factor contributing to mislocalization of TDP-43 in ALS/FTD.

Highlights

TDP-43 and FUS are ubiquitously expressed proteins that belong to the family of heterogenous nuclear ribonucleoproteins
nuclear export signal (NES)-239 (IAQSLCGEDLII) in TDP-43 and NES-289 (VQGLGENVTI) in FUS have been previously predicted as CRM1dependent NESs23,30, their activity has not been verified in bona fide nuclear export assays
Similar to the WT proteins, both single and double NES mutants of TDP-43 and FUS accumulated in mouse nuclei 2–5 hours post-fusion, whereas the non-shuttling control protein heterogenous nuclear ribonucleoproteins (hnRNPs)-C was only detectable in human nuclei (Fig. 1B)

Summary

Introduction

TDP-43 and FUS are ubiquitously expressed proteins that belong to the family of heterogenous nuclear ribonucleoproteins (hnRNPs) Their main site of localization is the nucleus, where they bind to gene promotors or long introns of pre-mRNAs and regulate transcription or splicing, respectively[3,4,5,6,7]. Inhibition of nuclear export as a therapeutic strategy has already been tested in preclinical models of C9orf72- and TDP-43-associated ALS and FTD: Here, specific inhibitors of the nuclear export receptor CRM1 (Exportin-1), KPT-276 and KPT-335, alleviated C9orf[72] repeat-mediated neurodegeneration in the drosophila eye[33] and reduced TDP-43 overexpression-induced cell death in cortical neurons[36], respectively. Functionality of the predicted NESs has not been tested in bona fide nuclear export assays

Methods

Results

Conclusion